Enzyme-Linked Immunosorbent Assay
A protein targeted method which uses an antibody-antigen reaction to determine if a specific protein is present. All proteins are extracted from the sample and then added to antibody-coated wells of a microtitre plate. Where the sample protein matches the antibodies on the plate, binding occurs. Non-matching proteins are removed through washing. A substrate is then added to effect a colour change which is readable with a spectrophotometer. The colour of the sample is converted to an optical density reading and measured in relation to the readings from a standard curve of known values. This enables us to quantify the levels of the target analyte present in the sample.
PCR – Polymerase Chain Reaction
A method of amplifying extracted DNA exponentially to create millions of copies of DNA. Uses timed sequences of heating and cooling, primers and polymerase to target and copy specific DNA sequences to enable detection.
Short strands of DNA unique to a single or group of species. Primers are designed for a specific target species or a generic species group. They are then used during PCR to copy the selected region of DNA from the sample (if present).
A test which will produce a value of presence if the analyte is detected. A range of quantitation usually exists which has a lower and upper limit. A result below the lower limit will be reported as ‘below limit of quantitation’ and is essentially not detected. A result above the upper limit will be reported as such and can be re-tested with a dilution to quantify the presence. A result within the range will be reported with a value relative to the total sample tested e.g. parts-per- million (ppm).
Real-Time PCR vs End-Point PCR
Real-time PCR records the progression of the polymerase chain reaction during the temperature cycles by detecting changes in fluorescence. Data is analysed on computer software by reading data curves on a graphical display.
End-point PCR is essentially the same as Real-Time PCR but the PCR product is analysed after the reaction. Gel electrophoresis separates the DNA within the product according to size and charge, and analysis is possible by reading the visible ‘bands’ under UV light.
A method of identifying the species from which DNA has been extracted. Greatly successful in pure samples (single species) but can be performed on most mixed samples if they produce a positive result for a specific target. Mainly used to identify the species where this is unknown or in question (for example to identify if a fillet of fish comes from cod or coley) and to confirm positive PCR results.
A process used in method validation to ensure the test can detect the target analyte if present, i.e. avoiding false negatives and also to ensure that the extraction is able to recover the analyte from the sample sufficiently. The ‘spike’ is a material with a known concentrate of target analyte (e.g. gluten). It is added to a sample with no detectable analyte (gluten-free) and the combined sample is then tested as normal.
A series of samples with known concentrations of analyte, which are included in each assay. These are used to generate a graphical curve along which the result of the sample may be read (if within range).
UOM – Uncertainty of measurement
Measurement of uncertainty is a mechanism used by analytical laboratories to attempt to identify how close the result obtained by the analyst is to the true result. It takes into consideration the normal variation of results produced by different analysts under different conditions, for example a sample may be tested on two separate occasions by two different analysts and give results of 19.5ppm and 20.5ppm. Both values fall within 95% of the average result – this is the confidence interval – and hence both results are valid. Calculating the uncertainty of measurement is key to interpreting results where critical limits are in place e.g. 20ppm for ‘gluten free’.